The field of the invention relates to spectrofluorometry. In particular, the field of the invention relates to adapters for cell holders of spectrofluorometers, wherein the adapter is configured to hold and position a microcell in the cell holder.
Fluorescence spectroscopy is one of the most sensitive methods for detecting biologically important molecules such as proteins, metabolites, and signaling molecules. In fluorescence spectroscopy, a light photon hits a molecule in an excitation process where the energy of the photon may be absorbed and re-emitted by the molecule through fluorescence. Often, the wavelength of the emitted photon is significantly longer than the wavelength of the excitation photon, which manifests itself as fluorescence in a different color. For example, excitation with blue light may cause green fluorescence. This phenomenon allows for sensitive detection of molecules capable of fluorescence emission (i.e., “fluorophores”) because the emitted light may be readily differentiated from the background, scattered excitation light on the basis of the wavelength of the fluorescence emission. Fluorescence measurements are used extensively in many laboratory and clinical assays. The instruments for fluorescence detection range from small benchtop devices specialized for specific analysis to large versatile setups that enable detection of multiple fluorophores in samples and analysis of the multiple fluorophores' properties.
Fluorescence measurement typically is performed in solution. In a typical fluorometric measurement, a liquid sample is placed in a quartz or glass cell, and the cell then is placed in a cell holder of a fluorometer. The light beam from an excitation source then is directed into the quartz or glass cell through a side wall to illuminate the liquid sample inside the cell, which fluoresces. Fluorescence is emitted approximately equally in all directions and, generally, is much weaker than the excitation light, which may reflect and scatter upon illuminating the liquid sample. Therefore, in order to acquire accurate fluorescence measurements, it is important to separate the fluorescence signal from the reflected and scattered light of the excitation source.
To achieve the best separation, the most common design of the high-sensitivity spectrofluorometer involves detection of fluorescence at a 90° angle relative to the direction of the excitation light beam. (See FIG. 1A and FIG. 1B). As illustrated in FIG. 1A, an excitation beam 6 enters through an excitation aperture 8 in an excitation screen 9a and contacts the illuminated area 4 of a cell 2. Fluorophores in a sample solution 16 contained in the cell 2 are excited and emit fluorescence 14 which is detected through the observed area 10 of the cell 2 as the fluorescence passes through the emission aperture 12 in an emission screen 9b to a detector. As illustrated in FIG. 1A and FIG. 1B, to reduce contribution from scattered light, the excitation light or beam 6 typically is “trimmed” using an excitation screen 9a having a narrow, rectangular or linear, excitation aperture 8 which permits the excitation beam 6 to illuminate only a middle area of the cell 4 and to prevent the excitation beam 6 from illuminating the corners of the cell 2. The same technique is used for detecting fluorescence where an emission screen 9b having a narrow, rectangular or linear, excitation aperture 12 permits the emission beam 14 to exit the middle of the cell 2 as an observed area 10 and enter the emission channel of a detector for detection. As a result, the fluorescence signal is detected exclusively from the middle of the sample cell, which is referred to as the “working volume” (see FIG. 1B), thus avoiding detecting any light (fluorescence or scattered) originating from the cell walls. As indicated in FIG. 1B, a “working volume” 18 of the sample solution 16 is contacted by the excitation light 6 as it enters through an excitation aperture in the cell holder 11. Fluorescence emission 14 from the working volume can pass only through the emission aperture in the cell holder 11. This approach to measuring fluorescence from soluble molecules is utilized in the majority of highly sensitive commercial spectrofluorometers.
Difficulties in detecting fluorescence may arise due to high optical density and sample turbidity. For example, difficulties regarding high optical density may result when the concentration of the fluorophore in the solution is high enough that the solution absorbs all of the excitation light before it has a chance to reach the “working volume” in the middle of the cell, a phenomenon termed “inner filtering effect.” The same difficulty may arise when turbidity in the sample due to suspended particles blocks the excitation beam. To enable observation of fluorescence in these situations, a conventional workaround involves illuminating the front surface of the cell and exposing the same surface to the emission channel. This workaround permits collection of the fluorescence signal from the portion of solution near the inner cell wall provided that the emission signal can be distinguished from the reflected and scattered excitation beam.
In recent years, some research has focused on the observation of fluorescence from solution-glass interfaces in order to investigate molecular structures absorbed on the glass surface (e.g., phospholipid bilayers). Most of these studies have been performed using horizontal glass slides where the solution drops are placed on top of the slide and observation are made from the bottom of the slide using fluorescence microscopes. The advantage of this setup is in its microscopic resolution of the surface details. However, the drawback to this setup is its poor accuracy in measurements of fluorescence intensity and, generally, its lack of spectral resolution in excitation and emission channels because of limitations of standard fluorescence microscopes. While it is possible to couple a fluorescence microscope with more sophisticated multi-wavelength excitation and emission channels, this is not a routine type of a fluorescence microscope and is extremely expensive to build.
One commercially available solution to enable observation of molecular layers absorbed on the inner glass surface of a sample cell using a standard horizontal-beam spectrofluorometer involves the use of triangular cells originally designed for measurements of samples with high optical density and/or turbidity. (See FIG. 2). Such cells are offered by the optical cell manufactures such as Starna Cells Inc. (See FIG. 3).
However, all of the cells that are currently manufactured have the front surface oriented at a 45 degree angle to the excitation channel, which creates a strong reflection of the excitation beam directly into the sensitive emission channel, effectively destroying signal-to-noise ratio of the measurement. Therefore, these triangular cells are not utilized for observation of molecular layers and are employed only in the analysis of turbid solutions when the fluorescence signal has a relatively high intensity.
Although unconventional cells having a non-45 degree angle relative to the excitation channel are not commercially available, such unconventional cells could be designed. However, an additional problem in using such unconventional cells would arise from the fact that the non-45-degree angle relative to the excitation beam would make the cells asymmetric such that the cell may be used only with the excitation beam coming from one specific side of the cell. This is problematic because many commercial spectrofluorometers have two excitation channels that illuminate the cell from opposite sides, for example, spectrofluorometers having a left channel equipped with a steady-state light source, and having a right channel equipped with a pulsed light source for time-domain measurements. Therefore, it would be impossible to perform steady-state and time-domain measurements on the same molecular sample using an unconventional cell having a non-45 degree angle. In this situation, the user would need to have two cells each made for the specific direction of the excitation light and create two identical samples in the two cells, which would complicate research design and increase costs.
Another important requirement for optical cells in biochemical research arises from the fact that sample quantities may be scarce for typical measurements. Therefore, there is a demand for so-called “microcells” such that the volume of the sample is within a microliter range (e.g., <100 μl). In order to utilize microcells in spectrofluorometers having 1-cm cell holders, adapters have been designed which center the microcell in the cell holder and provide reduced-size apertures for illumination and observation of the center of the sample solution while avoiding the cell corners (i.e., similar to illumination and emission screens).
Therefore, new microcell adapters for cell holders of spectrofluorometers are desirable. The new adapters preferably should receive and hold a microcell at a variable position (e.g., a variable skewed position) for detecting fluorescence of a sample at the inner surface of the microcell and permit use of the same microcell in spectrofluorometers that utilize multiple excitation channels for illuminating the microcell from opposite sides (e.g., from a right channel and from a left channel). The new adapters preferably should be configured for use in a commercially available spectrofluorometer having a 1-cm cell holder without having to modify the spectrofluorometer.